Non-simian cells for growth of porcine reproductive and respiratory syndrome (PRRS) virus

ABSTRACT

Disclosed are compositions and methods relating to growth of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using non-simian cells. In a particular example, porcine alveolar macrophage cells are described as having a capability of supporting infectivity and reproduction by PRRSV. Cells and cell lines of the invention are disclosed in connection with applications relating to PRRS disease, including vaccine technologies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/475,444 filed May 18, 2012, which is a divisional of U.S. applicationSer. No. 12/013,236 filed Jan. 11, 2008, which claims benefit ofapplication Ser. Nos. U.S. 60884782 filed Jan. 12, 2007 and U.S.60956597 filed Aug. 17, 2007, all of which are incorporated herein byreference to the extent not inconsistent herewith.

STATEMENT ON FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

BACKGROUND OF THE INVENTION

Porcine Reproductive and Respiratory Syndrome (PRRS) is a viral diseaseof swine that causes the greatest annual economic loss compared to anycurrent or previous infectious disease that has ever afflicted the porkindustry. PRRS, also known as “Mystery Swine Disease,” Swine Infertilityand Respiratory Syndrome (SIRS), and “Blue Ear Disease,” was firstdetected in North America in 1987 and in Europe in 1990. Since then,PRRS has since become a major threat to swine industries in mostpig-producing areas throughout the world, except Australia. PRRS has itsmost pronounced effect on young and newborn piglets. Up to 20-30% of thepiglets in the litters from infected sows are stillborn, and up to 80%of piglets in infected herds die before weaning. The economicconsequences of the disease, accordingly, are devastating (see U.S. Pat.No. 5,476,778 by Chladek et al.). In a study funded in part by theNational Pork Board, it was noted that losses attributed to PRRSexceeded $560M in the United States alone (Neumann E J et al., 2005).

The research of PRRS and development of diagnostic and therapeuticapproaches, including new vaccines and vaccine production technology, isconstrained by the restricted availability of options for culturing thePRRS virus in vitro. Previously, simian cells have been the only type ofavailable cell line known to sustain growth of PRRS for vaccineproduction. The simian cell line MA-104 and related derivatives (e.g.,MARC-145) may represent what until now might have been the onlypractical option for supporting PRRS virus replication in vitro.

The present invention improves the state of the art by providingalternative options for working with PRRS virus such as the embodimentof non-simian cells that can support PRRS growth.

Other approaches have been taken, with less than desirable outcomes, todevelop options for growth of PRRSV. In one approach, porcine alveolarmacrophages can be used for PRRSV growth; however, the high degree ofinherent variability in cells isolated from different pigs is considereda disadvantage (see Vincent et al., 2005; Bautista et al., 1993). Inanother approach, Weingartl et al. established porcine monomyeloid celllines following transfection of primary porcine alveolar macrophagesobtained from 12-week-old pigs. Those cells were tested but failed tosupport replication of PRRSV (Weingartl et al., 2002, Journal ofVirological Methods 104:203-216). In contrast the present inventionsurprisingly discloses, inter alia, the discovery that certain cellsfrom fetal pigs are indeed capable of supporting PRRSV growth.

Technology applications of great interest for utilization of the presentinvention include applied research aspects such as veterinary vaccineproduction in addition to the ability to study the growth of the PRRSvirus at the basic research level. Vaccines for PRRSV which are modifiedlive vaccines or inactivated killed/vaccines can be produced by thecells and cell lines of the invention. A tool to grow this particularvirus and related methods also are significant in the context ofgenerating material useful for diagnostic applications. In the contextof a historical analogy intended to emphasize breakthrough science, theability to work with human immunodeficiency virus (HIV) was generallystifled due to difficulties and a lack of options for culturing thevirus in vitro. Crucial advances in the ability to study and generatediagnostics and potential vaccines for HIV related to the watershedevent of being able to successfully culture the virus in vitro; seeGallo R C, 2002, Historical essay. The early years of HIV/AIDS; Science298(5599):1728-30.

The major limitation to the control of PRRS disease is believed to restwith the availability and efficacy of vaccine technologies. Thecontribution of innovative cells and methods for growth of PRRS virusthus represents a significant advance in the ability to work with PRRSvirus, to develop alternative and/or improved vaccine technologies, andto address the problem of PRRS disease.

SUMMARY OF THE INVENTION

The invention broadly relates to non-simian cells capable of reproducingPRRS virus and methods for reproducing PRRS virus using such cells.

In an embodiment, the invention provides an isolated non-simian cellcapable of reproducing the PRRS virus, wherein the non-simian cell isobtained from an individual animal and cultured for at least 5 passages.

In an embodiment, the invention provides an isolated porcine fetal lungcell capable of infection by and/or reproducing the PRRS virus. In anembodiment, the cell is an alveolar macrophage cell. In an embodiment,the cell is propagated in culture for at least 5 passages. In anembodiment, the cell is propagated in culture for at least 10 passages.In an embodiment, the cell is propagated in culture for at least 20and/or 50 passages.

In an embodiment, the invention provides an isolated primary cell orcell population obtained from a lung of a porcine fetus. In anembodiment, the fetus is from about 30 to about 90 days of gestationalage. In an embodiment, the cell is an alveolar macrophage or the cellpopulation comprises at least one alveolar macrophage cell. In anembodiment, the cell is a macrophage, of macrophage lineage, orprogenitor thereof.

In an embodiment, the invention provides a cell or cell line designatedherein as ZMAC. In an embodiment, the invention provides a cell or cellline designated herein as FBAL-A. In an embodiment, the inventionprovides a cell represented by a deposit with the American Type CultureCollection designated as ATCC Patent Deposit No. PTA-8764 or derivedtherefrom.

In an embodiment, the invention provides an immortalized cell variant orderivative of a cell described herein having the capability ofsupporting PRRSV growth.

In an embodiment, the invention provides a method of generating progenyof a PRRS virus comprising:

a) providing an isolated or purified non-simian cell capable ofreplicating a PRRS virus, wherein said non-simian cell is cultured forat least 5 passages;

b) exposing said non-simian cell to said PRRS virus; and

c) allowing the PRRS virus to replicate in the cell;

thereby generating progeny of a PRRS virus. In an embodiment, thenon-simian cell is obtained from an individual animal. In an embodiment,the method further comprises harvesting the grown PRRSV.

In an embodiment, the invention provides a method of producing a PRRSvaccine, comprising providing a modified-live virus (MLV) strain ofPRRSV, and growing said MLV strain in an isolated or purified non-simiancell capable of replicating said PRRSV MLV strain. In an embodiment, thenon-simian cell is a macrophage cell or of macrophage lineage or aprogenitor thereof. In an embodiment, the non-simian cell is a porcinecell or derivative thereof. In an embodiment, the non-simian cell is aporcine alveolar macrophage cell. In an embodiment, the non-simian cellis a porcine alveolar macrophage primary cell, cell population, variantor derivative thereof. In an embodiment of the method, the cell isrepresented by a cell designated as ZMAC-1, FBAL-A, or a deposit withthe American Type Culture Collection designated as ATCC Patent DepositNo. PTA-8764 or derived therefrom.

In an embodiment, the invention provides a composition comprising cellor cell line designated as ZMAC-1 or represented by a deposit with theAmerican Type Culture Collection designated as ATCC Patent Deposit No.PTA-8764 or derived therefrom. In an embodiment, the invention providesa composition comprising a cell or cell line designated as FBAL-A.

In an embodiment, the invention provides a method of isolating a cellfrom a porcine fetal lung comprising providing a porcine fetal subject;obtaining a cell-containing bronchoalveolar lavage sample from saidsubject; and separating a cellular component from said sample; therebyisolating said cell. In an embodiment, said cell-containing sample is abronchoalveolar lavage sample. In an embodiment, said sample is anexcised tissue sample which is optionally processed by maceration and/orhomogenization.

In an embodiment, the invention provides a method of growing a virus,comprising: (a) isolating a cell from a porcine fetal lung; (b)culturing said cell; and (c) contacting said cell with said virus so asto allow viral replication; thereby growing the virus. In an embodiment,said virus is a PRRS virus. In an embodiment, said virus is anattenuated PRRS virus or a PRRS virus field isolate. In an embodiment,the field isolate is virulent or naturally of low virulence ornon-virulent. In an embodiment, said culturing comprises passaging saidcell for at least 5 passages and/or growing said cell for at least 10days of continuous culture.

In an embodiment, the invention provides a method of growing andisolating PRRSV, which comprises inoculating the virus on a culture ofnon-simian cells in the presence of serum in a suitable growth mediumand incubating the inoculated cells until PRRSV progeny virus materialis generated. In a particular embodiment, the PRRSV is ATCC-VR2332. In aparticular embodiment, the non-simian cells have been previously grownin culture for at least 5 passages. In an other particular embodiment,the non-simian cells have been previously cultured for at least 10, 20,and/or 50 passages.

In an embodiment, the invention provides a method of growing PRRSVcomprising (a) inoculating PRRSV on non-simian cells that have beenpreviously cultured for at least 5, 10, 20, and/or 50 passages; and (b)incubating the inoculated non-simian cells. In an embodiment, thenon-simian cells are porcine alveolar macrophages. In an embodiment, thenon-simian cells are obtained from a porcine fetal lung sample. In anembodiment, the PRRSV is derived from a homogenate of swine tissueinfected with the virus. In an embodiment, the method comprisesincubating the culture until a fixed time period and/or until acytopathic effect is observed. In an embodiment, the fixed time periodis from about 16 to about 72 hours.

In an embodiment, the invention provides an immunogenic compositioncomprising inactivated PRRSV, wherein the inactivated virus is formed bya process which includes growing PRRSV on non-simian cells prior toinactivating the virus, wherein the non-simian cells have beenpreviously cultured for at least 5, 10, 20, and/or 50 passages. In anembodiment, growing the virus comprises: (a) inoculating swineinfertility and respiratory syndrome virus on the cells; and (b)incubating the inoculated cells at about 34 to about 37 degrees C. In anembodiment, growing the virus involves a growth medium including serum.In an embodiment, the inactivated virus is killed PRRSV strain ATCCVR-2332. In an embodiment, the immunogenic composition further comprisesan adjuvant and/or a pharmaceutically acceptable carrier.

In an embodiment, the invention provides a composition comprising avirus grown in a cell of the invention. In an embodiment, the virus is aPRRS virus (which can include a virus derived from a PRRS virus).

In an embodiment of methods of the invention, a cell of the invention iscontacted with a growth factor composition. In an embodiment, the growthfactor composition comprises macrophage colony stimulating factor(MCSF). In an embodiment, the growth factor composition comprisesgranulocyte-macrophage colony stimulating factor (GMCSF).

In an embodiment, the invention provides a composition comprising PRRSVin or from a culture of non-simian cells; wherein the non-simian cellshave been previously cultured for at least 5, 10, 20, and/or 50passages, are derived from a porcine alveolar macrophage originatingfrom a fetal porcine lung sample, or both. In an embodiment, the PRRSVis a fastidious, non-hemagglutinating, enveloped RNA virus and iscapable of effecting PRRS in swine. In an embodiment, the compositionhas a virus titer of about 10³ to about 10⁷ TCID₅₀/ml. In an embodiment,the virus titer is up to about 10⁹ TCID₅₀/ml. In an embodiment, thePRRSV is an MLV strain. In an embodiment, the PRRSV is derived from aninoculum comprising a homogenate of tissue from a swine affected withPRRS. In an embodiment, the inoculum is derived from a neutralizedtissue homogenate, and the neutralized tissue homogenate is obtained byneutralizing the tissue homogenate with antibody sera to swine diseasesselected from the group consisting of hemophilus, brucellosis,leptospira, parvovirus, pseudorabies, encephalomyocarditis, enterovirus,swine influenza, and mixtures thereof. In an embodiment, the inoculum isderived from a filtered homogenate of the tissue homogenate, thefiltered homogenate containing particles having a size no greater thanabout 0.45 micron.

In an embodiment, the invention provides a non-simian cell wherein thecell is a porcine cell. In a particular embodiment, the cell is derivedfrom a porcine lung. In a preferred embodiment, the cell is derived froma porcine fetal lung. In a particular embodiment, the porcine fetal lungis from a porcine fetus of a gestational age from about 20 to about 80days. In a particularly preferred embodiment, the porcine fetus has agestational age of from about 50 to about 70 days. In an embodiment, thecell is obtained from a lung lavage sample from a porcine fetus. In aparticular embodiment, the cell is a porcine alveolar macrophage cell.

In an embodiment, nonadherent and/or relatively less adherent cells arepreferably selected. In an embodiment, a mixed cell population isobtained from a porcine fetal lung. In an embodiment, there are at leasttwo cell types in the mixed cell population including a precursor cellof macrophage/monocyte lineage which is self-renewing and relativelyless differentiated or undifferentiated; and a differentiated cell ofmacrophage/monocyte lineage which is relatively more differentiated. Inan embodiment, there is at least a third cell type in the mixed cellpopulation which is a feeder/helper cell type. In an embodiment, thethird cell type is either from an endogenous sample or is addedexogenously.

In an embodiment, a cell and/or cell population of the invention can bedescribed as having a population of precursors of endothelial cells thatcan be made to produce endothelial cells under appropriate cultureconditions. For example, the culture conditions can include the type ofplasticware and/or surface treatments.

In an embodiment, there can be a fraction of cells that express CD90 andCD117. In a particular embodiment, the fraction is about 30% or at least30%.

In an embodiment, a non-simian cell of the invention is used to producea vaccine against Porcine Reproductive and Respiratory Syndrome Virus(PRRSV).

In an embodiment, a cell or cell line of the invention is capable ofsupporting growth of a PRRS virus which is a wild-type virus or alaboratory strain. In a preferred embodiment the invention supportsgrowth of a PRRS modified live virus. In a particular embodiment, thevirus is a strain corresponding to or derived from a BoehringerIngelheim product lines such as Ingelvac® PRRS and ReproCyc® PRRS swinevaccines. In a particular embodiment, the virus is a straincorresponding to or derived from a Schering-Ploug product line ofPrime-Pac® PRRS vaccine. In a particular embodiment, the virus is anisolate used in United States Veterinary Biological Product License(Mar. 29, 1996) for Porcine Reproductive and Respiratory SyndromeVaccine, Reproductive Form, Killed Virus, Code 19S5.20. In anembodiment, the PRRS virus is an attenuated virus. In an embodiment, thePRRS virus is a field isolate. In an embodiment, the field isolate is avirulent strain or a strain of naturally low virulence or non-virulent.In an embodiment, compositions or methods of the invention areapplicable for growth of PRRS virus material which is used formanufacture of live, attenuated, or inactivated vaccine.

In an embodiment of a cell or cell line of the invention, animmortalized derivative is established from starting material of anon-immortalized or already immortalized cell or cell line. In anembodiment, an immortalized cell line or cell line is established usingone or more transformation or other immortalization techniques as isunderstood in the art.

In an embodiment of a cell or cell line of the invention, one or moresubclones are established. For example, a subclone is isolated andreproduced according to conventional techniques such as by limitingdilution in culture.

In an embodiment of the invention, a primary cell or cell line iscultured for at least 5 passages. In other embodiments, a primary cellor cell line is cultured for at least 10 passages, at least 20 passages,and/or at least 50 passages. In an embodiment of the invention, aprimary cell or cell line is cultured for at least one week. In anembodiment of the invention, a primary cell or cell line is cultured forat least two weeks. In other embodiments, a primary cell or cell line iscultured for at least four weeks, at least eight weeks, and/or at leastsixteen weeks.

In an embodiment, a composition such as a cell or cell line is isolatedor purified.

In an embodiment, the invention provides a process for growing a porcinereproductive and respiratory syndrome virus (PRRSV) by growing the virusin a tissue culture to an amount sufficient to protect animals againstPRRS to diagnose PRRS or to identify the molecular structure of PRRSVfor subunit or recombinant products, comprising inoculating PRRSV onto atissue culture of a non-simian cell or cell line and harvesting thegrown virus. In an embodiment, the cell or cell line is porcine. In anembodiment, the cell or cell line is a porcine alveolar macrophage. Inan embodiment, the invention provides a tissue culture containing thePRRSV produced according to the process. In an embodiment, the inventionprovides a process for preparing an effective vaccine for protectingpigs against PRRS comprising providing PRRSV as described herein,releasing the PRRSV from the tissue culture cells and adjustingantigenic mass by dilution, concentration or extraction to produce animmunologically effective amount of the antigenic mass for a relativelyintact, subunit, or recombinant product.

In an embodiment, a non-simian cell or cell line of the invention isused to produce live PRRSV or inactivated/killed PRRSV.

In an embodiment, the invention provides a purified non-simian cell orcell line of macrophage lineage derived from a fetal porcine organism.In an embodiment, the invention provides a method of generating apurified non-simian cell or cell line of macrophage lineage derived froma fetal porcine organism. In an embodiment, the cell or cell line iscultured in vitro for at least three, five, or 10 passages.

Without wishing to be bound by any particular theory, there can bediscussion herein of beliefs or understandings of underlying principlesor mechanisms relating to the invention. It is recognized thatregardless of the ultimate correctness of any explanation or hypothesis,an embodiment of the invention can nonetheless be operative and useful.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the growth of two PRRSV attenuated strains (PrimePacand Syva) in a culture of non-simian cell UIMAC FBAL-A.

FIG. 2 illustrates the phenotype of UIMAC-FBAL-A cells including theexpression of characteristic macrophage cell surface markers.

FIG. 3 illustrates morphological features of FBAL-A cells usingmicroscopy techniques: A) Giemsa stain to show morphological appearance(cytospin); B) Confocal microscopy of FBAL-A stained for CD14; C)Confocal fluorescence microscopy staining for CD172; and D) Staining ofFBAL-A infected with PRRSV staining for nucleocapside antigen whichindicates a characteristic localization of N antigen to nucleus andassociation with nucleolus. FIG. 3B and FIG. 3C illustrate the actualmorphology of fresh cells stained for CD14 or CD172 with characteristicfilopodia and lamellipodia.

FIG. 4 illustrates data on growth kinetics of the ZMAC cells (FIG. 4A)and PPRSV growth using the cells (FIG. 4B).

FIG. 5 illustrates ZMAC cells at 20 hours after being infected with PRRSvirus strain NADC20 at MOI of 1 (FIG. 5A) and 0.1 (FIG. 5B). Cells werefixed and stained with FITC labeled SDOW17 mAb that is specific for thenucleocapsid (N) protein.

FIG. 6 illustrates results of weight change in pigs after exposure towild-type PRRS virus.

FIG. 7 illustrates the extent and frequency of viremia in pigs afterexposure to wild-type PRRS virus.

FIG. 8 illustrates results of measuring virus load in samples from thebronchalveolar lavage of pigs after exposure to wild-type PRRS virus.

FIG. 9 illustrates growth of ZMAC-1 cells in the presence of growthfactors.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are applicable: PRRS, Porcine Reproductiveand Respiratory Syndrome; PRRSV, PRRS Virus; TCID₅₀, tissue cultureinfectious dose 50% level; ATCC, American Type Culture Collection; MOI,multiplicity of infection.

In general the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in the context ofthe invention.

When used herein, the term “cell” can refer to a biological entity aswould be understood in the art and which is intended to encompassspecific entities that may be described as a primary cell or a cellline. When several of these terms are used herein, it will beappreciated by one of ordinary skill that such usage is merely forpurposes of emphasizing well understood distinctions. For example, thephrase “a cell or cell line” may emphasize the contrast between anoriginal primary isolate versus an immortalized version which could be adirect derivative of the original primary isolate.

When used herein, the term “isolated” refers to a manipulated state thatis different than that which is the natural state and/or is modifiedrelative to a starting material, in which case the term is meant to beconsistent with the concept of being purified. For example, an isolatedprimary cell is excised from a natural tissue or other source in a hostorganism and maintained apart from the original source. As anotherexample, a cell component can be placed in culture or further separatedfrom a lung lavage fluid-based sample, thus achieving a relativelyisolated cell.

When used herein, the term “purified” refers to a condition whereinthere has been a relative enrichment, separation, and/or removal of asubstance relative to a starting material. The term can encompassconditions of an at least partial purification and does not necessarilyimply an absolute state of purity. For example, the term can apply to acell that is capable of reproducing a PRRS virus and present in a mixedculture and independently can be applicable to what may customarily beconsidered a pure culture. The term can apply to a primary cell culturewhich is optionally a mixed culture. The term can apply to a cell line.

In an embodiment, the invention provides a porcine alveolar macrophagecell line which is capable of supporting the growth of PRRSV. Anexemplary composition of the invention is a non-simian cell line.Particular compositions of the invention include a cell or cellpopulation obtained from porcine fetal lung samples. A further specificcomposition includes a fetal porcine alveolar macrophage. In anembodiment, the invention provides methods of growing PRRSV in anon-simian cell or cell line.

The invention may be further understood by the following non-limitingexamples.

EXAMPLE 1 The Non-Simian Cell Isolate UIMAC FBAL-A is Capable ofSupporting PRRSV Growth

The porcine alveolar macrophage cell line UIMAC FBAL-A was derived fromthe lung of a 45 day old porcine fetus by bronchoalveolar lavage withculture medium. The initial culture was expanded in vitro from theinitial few thousands of cells to several million over a period of 6months. At that time, experiments were conducted to measure the growthof PRRS virus. These experiments showed that the FBAL-A cells were ableto support the replication of the virus. This isolate was notdeliberately transformed yet demonstrated an ability to grow andpropagate in culture and a phenotype for the ability to supportinfectivity and growth of PRRS virus.

The cell culture conditions were as follows. Culture medium: RPMI 1640supplemented with L-glutamine and 25 mM HEPES. The medium issupplemented with non-essential amino acids, sodium pyruvate,gentamicin, and 10% fetal bovine serum. Cells were incubated at 37° C.with 5% CO₂ at 100% humidity. Tissue culture grade 6 well plates wereused for the culture surface. A culture volume of 4-6 ml of culturemedium/well was used. For passages, cells were selected for beingnon-adherent or weakly adherent. Every 3-4 days a portion of the cellswere removed from the culture with the aid of a Pasteur pipette and arubber bulb by gentle suspending the cells in the culture fluid andtransferring ⅓ of the well volume to a new well. The new well and thesource well were fed with fresh medium.

FIG. 1 illustrates the growth of PRRSV attenuated strains PrimePac andSyva in UIMAC FBAL-A. The results demonstrate that the porcine cells canbe used to produce PRRS vaccine virus in quantities sufficient forvaccine production.

The isolated porcine cells can be identified and/or isolated byexpression of certain surface molecules. The surface molecule pattern ofexpression can be used to isolate other independent porcine cells or toscreen cell lines as potential indicators for the capability ofsupporting PRRSV growth. For instance, cells are optionally screened forCD163 expression. In a particular example, cells or cell lines aresubjected to staining and/or separation techniques by virtue of cellsurface molecules.

FIG. 2 illustrates the phenotype of UIMAC-FBAL-A cells including theexpression of characteristic macrophage cell surface markers. The FBAL-Acells express CD14, CD163, and CD172. The protein molecule CD163 isindicative of monocyte/macrophage lineage and correlates withpermissiveness of infection by African swine fever virus. The cells alsoexpress MHC class II molecules at low level. The red line towards theleft side represents the staining results of the negative controlsample. It is noted that expression of certain marker, including suchdescribed herein, can be used for characterization of the cells, butindependently can also be used for enrichment, separation (e.g., bysorting), and other purposes. In an embodiment, a cell such as amacrophage cell of the invention may not necessarily express one or moregiven certain markers, or its expression pattern may change over time,but may nonetheless be capable of replicating PRRSV.

FIG. 3 illustrates morphological features of FBAL-A cells usingmicroscopy techniques: A) Giemsa stain to show morphological appearance(cytospin); B) Confocal microscopy of FBAL-A stained for CD14; C)Confocal fluorescence microscopy staining for CD172; and D) Staining ofFBAL-A infected with PRRSV staining for nucleocapside antigen whichindicates a characteristic localization of N antigen to nucleus andassociation with nucleolus. FIG. 3B and FIG. 3C illustrate the actualmorphology of fresh cells stained for CD14 or CD172 with characteristicfilopodia and lamellipodia.

In microscopic examination experiments, cells were centrifuged onto aslide and stained with Giemsa stain. Unfixed cells were incubated withmonoclonal antibody specific for SWC3 or CD14 followed by exposure toFITC-labeled goat anti-mouse Ig. For PRRSV nucleocapsid detection, cellswere infected with PRRS virus (strain VR-2332). After 18 h, the cellswere centrifuged onto a slide, fixed with acetone, and incubated withFITC-labeled anti-PRRS virus N protein mAb SDOW17. Uninfected cells didnot react with mAb SDOW17. In growth kinetic studies, PRRSV isolateVR2332 was used in UIMAC-FBAL-A cells. Virus yields obtained from themacrophage cell line were titrated in MARC-145 cells.

Without wishing to be bound by theoretical explanation, for certaincompositions it is believed that cells described herein derived fromporcine fetal lungs can contain self-regenerating progenitors. Theseself-regenerating progenitors can produce progeny macrophages, which canfurther differentiate, and moreover can have the ability to produceadditional progenitor cells allowing continuing propagation.

EXAMPLE 2 The Non-Simian Cell Isolate ZMAC is Capable of SupportingPRRSV Growth

In addition to the development of FBAL-A cells, an independent effortresulted in the development of a second cell isolate designated as ZMAC.ZMAC cells were characterized and observed to have the capability ofsupporting PRRSV infectivity and growth.

In this independent attempt, six 58 day-old fetuses were asepticallyharvested from the uterus of sow 9688 obtained from the swine herd ofthe University of Illinois Veterinary Medicine Research Farm. This sowwas a cross-bred pig with the following breed composition: 17/32Landrace, 13/32 Yorkshire and 2/32 Duroc. The fetuses were transportedto the cell culture laboratory and their lungs dissected inside of abio-safety cabinet under sterile conditions.

Cells in the airways of the lungs from each of the 6 fetuses wereisolated separately by bronchoalveolar lavage. Approximately 100,000cells were obtained from the lung of each fetus. The cells from eachfetus were cultured separately in different wells of a 6-well tissueculture plate. After an initial 4-day culture, the cells were purifiedvia a Ficoll-Hypaque density gradient. In an embodiment, cells can bepurified by Ficoll-Hypaque either the day of isolation or at a latertime, e.g., 1, 2, or 3 weeks later, it is recognized that this canfacilitate removal of certain components such as red cells, andpotentially other material, present in an original preparation). Within20 days after establishment of the initial cultures, the cells wereexhibiting robust growth and were split into additional 6-well plates.These cells were named ZMAC/FBAL-II and further designated as sublines1-6 to identify the cells isolated from the 6 different fetuses.Subsequently the cells were split approximately every 4-5 days. Therobust growth of cells was evident by the presence of cell clusterswhich started to double in terms of cells per cluster about every 2.5 to3 days. By 60 days after the initiation of the culture, theZMAC/FBAL-II.1, and ZMAC/FBAL-II.2 cells exhibited better growth thanthe other four lines. Upon further passages, the ZMAC/FBAL-II.1, andZMAC/FBAL-II.2 lines were growing sufficiently well to be transferred to75 cm² tissue culture flasks. Culture conditions were generally asdescribed above. In this experiment, Sarstedt flasks for suspensioncultures were used.

Upon multiple passages, the cells exhibited vigorous growth; severalmillion cells were able to be harvested every 10-12 days. Growth curvesindicated that the cells are capable of replicating PRRS virus as wellas the FBAL-1 (FBAL-A) cells. In one assay, the yield of virus wasapproximately 0.2 TCID₅₀/cell. Two types of modified live PRRS virusisolates were tested for growth in these cells (PrimePac strain ofSchering-Plough and the Spanish vaccine isolate of Syva in Europe).

Five lots of cells were frozen and stored in liquid nitrogen. Each lothas a minimum of 10 vials with two million cells each. A representativevial from each lot was found to have >80% viability after being thawedand to exhibit vigorous growth within 4 days after culture initiation.The ZMAC population has been found to be 70% susceptible to infectionwith PRRS virus as determined immunofluorescence staining for viralproteins within 18 hours after infection at an MOI of 0.01. In anotherassessment, we determined that the ZMAC cell line is 100% susceptible toinfection by PRRS virus, as determined by immunofluorescence stainingfor viral proteins 12 hours after infection at an MOI of 10.

We have also performed multiple step growth curves by infecting the ZMACcells with PRRS virus (PrimePac) at an MOI of 0.02. Based on real timePCR analysis of virus genome expression in the infected cells and bytitrating the amount of virus progeny produced, we have determined thatthe first round of replication of PRRS virus is completed by 9 hoursafter infection, and that the peak yield of virus progeny is achieved bythe second round of replication at 18 hours after infection (FIG. 4B).

In FIG. 4, FIG. 4A shows results of growth kinetics of the ZMAC cells.Established cultures of ZMAC cells were grown in 75 cm² culture flaskscontaining 15 ml of cells at 2-3.2×10⁵ cells/ml; cells were fed with anequal volume of fresh culture medium to achieve a cell density of1-1.6×10⁵ cells/ml. Cells were counted at 0, 3, and 6 days after freshmedium was provided. Each flask is capable of producing 2-3×10⁶ cellsevery four days.

FIG. 4B illustrates a multiple step growth curve of PRRS virus on ZMACcells. In these experiments, the results indicate the viral yields andkinetics of PRRSV growth on ZMAC cells. A suspension of ZMAC cells at1×10⁶/ml were infected at an MOI of 0.02 with attenuated PRRS virusisolate PrimePac. After one-hour incubation at 37 C. the inoculum wasremoved by centrifugation, and the cells were suspended at 1×10⁶/ml. A0.1 ml volume was removed at the indicated times after infection and thenumber of TCID₅₀ units determined in MARC-145 cells. The attenuated PRRSvirus strain PrimePac, potentially suitable as a vaccine material, growswell in the ZMAC cell line.

FIG. 5 illustrates ZMAC cells at 20 hours after being infected with PRRSvirus strain NADC20 at an MOI of 1 (FIG. 5A) and 0.1 (FIG. 5B). Anegative control with mock infected cells was also observed (not shown).Cells were fixed and stained with FITC labeled SDOW17 mAb that isspecific for the nucleocapsid (N) protein. Phase contrast microscopy wasalso performed to observe the cells. Generally these cells do not showCPE until about 28-36 hours after infection. A substantial amount ofvirus material, however, is released from the cells much earlier, forexample by about 18-20 hours after infection.

Samples of cells are used to adapt cell growth conditions and viralgrowth conditions for purposes of maximizing and/or optimizing virustiters. PRRS viral stocks are generated accordingly and can be used forvaccine development and vaccination purposes.

EXAMPLE 3 Immortalized Non-Simian Cell Lines Capable of Supporting PRRSVGrowth

A cell or cell line of the invention is established as one or more of aspontaneous immortalized variant, deliberately transformed derivative,other variant or derivative, and an otherwise immortalized version of aprimary cell or cell line as described herein.

For instance, techniques are utilized as would be understood in the art,such as involving viral transformation technology; fusion with animmortalized partner; exposure to chemical/physical conditionsincluding; e.g., irradiation; and technology relating to manipulation oftelomerase function. In a preferred approach, the tert system is used toestablish a cell or cell line which can avoid, delay, or otherwise altersenescence. See, e.g., Carrillo et al., Veterinary Immunology andImmunopathology 89 (2002) 91-98; Kwak S et al., 2006 AnimalBiotechnology, 17: 51-58;http://www.atcc.org/common/products/CellImmortProducts.cfm.

In preliminary isolation efforts or downstream development of animmortalized cell, aspects such as purification and/or subcloning arefacilitated by fluorescence activated cell sorting or other flowcytometry separation or analysis technique. Other techniques such asmagnetic cell separation are adaptable for use with cells and methodsherein.

EXAMPLE 4 Determination of Immunogenicity and Efficacy of a PRRSModified Live Vaccine

In this example, the immunogenicity and efficacy of a PRRS modified livevirus vaccine produced using a porcine macrophage cell line isdetermined. A porcine alveolar macrophage cell line is tested. In aparticular instance, the porcine macrophage primary cell linesUIMAC-FBAL-A and/or ZMAC are used. Alternatively, one can generateindependent starting materials and/or derivatives such as transformantsaccording to the disclosure herein.

A PRRS MLV candidate is selected and produced in connection with a cellor cell line as described herein to generate PRRS MLV material forclinical trial evaluation. A vaccination and challenge study isconducted with 32 pigs that are four weeks of age, randomly allocatedinto four groups of n=8 per group. The pigs are obtained from theUniversity of Illinois Veterinary Research Farm PRRS virus-free swineherd and acclimated at an isolation facility for one week beforestarting the study.

Animals in groups 1 and 2 are immunized once in the rump area with adose of 5×10⁴ TCID₅₀ units in a 2 ml volume of the MLV stock grown ineither the UIMAC-FBAL-A cells or in MARC-145 cells. Animals in group 3are injected with a 50:50 mixture of 2 ml of spent culture supernatantfrom UIMAC-FBAL-A and simian MARC-145 cells. The animals in group 4 arenot treated and serve as controls. As a measurement of virus-specificimmunity, peripheral blood samples are collected from each animal atweeks 0, 2, 4, 6 and 8 weeks post-immunization. From these samples,serum and PBMC are obtained and the intensity of the humoral andcell-mediated immune responses measured. The titer of serumvirus-neutralizing antibodies and the frequency of PRRS virus-specificinterferon (IFN)-gamma-secreting sells (SC) separately against the twoparental PRRS virus isolates FL-12 and NEB-1, a chimeric virus (Kwon B,2006), and virulent challenge strain VR2385 (Opriessnig T, 2002). Tomeasure the level of protective immunity induced by the vaccinematerial, animals in groups 1-3 are challenged at 6 weekspost-immunization by instillation of 2 ml of the virulent PRRS virusstrain VR2385 (10⁵ TCID₅₀ doses per 2 ml, administered in aliquots of 1ml per nostril). Animals in group 4 remain unvaccinated and are notchallenged in order to determine normal clinical parameters as well asnormal lung appearance. The PRRS virus-naïve animals in group 3 willserve as the challenge controls to determine the severity of thechallenge. The degree of protective immunity elicited by the vaccine isestablished based on factors including the measurement of bodytemperature and weight change and the observance of clinical signs suchas depression and respiratory signs. These parameters are monitoreddaily for fourteen days. The level of viremia, IFN-gamma response, andbody weight are determined at 0, 4, 7, 10 and 14 days post-challenge.Fourteen days after challenge, the animals are euthanized, and the viralload in lung tissue and bronchoalveolar lavage fluid determined.Pathological changes in the lung are assessed at the macro andmicroscopic levels.

In another experiment, the immunogenicity and efficacy of biologicmaterial generated from ZMAC is evaluated. The ZMAC material is comparedto the same vaccine virus but grown in the MARC-145 cells. For thispurpose we have performed 3 serial passages of the PrimePac virus strainin ZMAC cells. The third passage of this virus in the ZMAC cells isutilized to conduct the comparative vaccination study of this biologicto the PrimePac virus grown in MARC-145 cells. Stocks of the PrimePacvaccine grown in either the ZMAC or MARC-145 cells are prepared to atiter of 10⁶ TCID₅₀/ml. Groups of pigs are immunized at 5×10⁴ TCID50dose in a 2 ml volume of the MLV stock grown in either the UIMAC-FBAL-Acells or in MARC-145 cells.

EXAMPLE 5 Use of a Porcine Alveolar Macrophage Cell Line to Produce aPRRS Modified Live Virus Vaccine

Introduction. In mid-1994, the first PRRS MLV vaccine (Ingelvac PRRSMLV) was released. Since then, the use of attenuated viruses as vaccineshas become customary in North America and Europe. It is well acceptedthat these agents are effective in conferring appropriate levels ofhomologous protective immunity while affording variable degrees ofprotection against challenge by heterologous strains (Mengeling et al.,1996; Mengeling et al., 1999). Thus there is motivation and interestdirected for further progress in technologies of alternative andimproved strategies, vaccines, and tools and methods in connectiontherewith.

In this study, a porcine alveolar macrophage cell line, designatedZMAC-1, is used for the production of a PRRS modified live virus (MLV)vaccine. Also, the efficacy of vaccine virus grown in this porcine hostis compared to that propagated in the only other cell line known at theinitiation of this study to support the growth of PRRS virus, namely thesimian cell line MA-104 and/or its derivative the MARC-145 line.

Initially, the ZMAC-1 cells were found to be readily susceptible to theMLV vaccine Prime Pac PRRS (Schering-Plough Animal Health). Moreover,after the third passage of the virus in ZMAC-1 cells, a yield comparableto that achieved when using MARC-145 cells was obtained. To evaluate thevaccine potential of both ZMAC-1 cell-grown virus, a standardimmunization-challenge study was conducted. In this case, six 8 week-oldpigs were injected with an equivalent dose of the Prime Pac vaccinegrown in either ZMAC-1 or MARC-145 cells while two additional groups ofthree animals were not immunized. Four weeks later, all vaccinated andone of the PRRS virus-naïve groups were challenged with the “atypicalPRRS abortion storm” virus isolate NADC-20. One outcome of the study wasthat the Prime Pac MLV vaccine grown in either cell line was equallyeffective at preventing the body weight loss of PRRS virus-naïve pigsthat had been exposed to the heterologous virus 7 days earlier. However,the vaccine virus grown in ZMAC-1 cells was significantly more effectivethan that grown in MARC-145 at reducing the extent of viremia and alsoat eliminating virulent virus from the lungs at 7 and 10 dayspost-challenge, respectively.

The observation that the type of cell line used to grow the PRRS MLVvaccine can improve the level of protective immunity elicited by thesame vaccine virus against a genetically divergent virulent PRRS virushas important implications for the prospect of developing a highlyeffective vaccine against this pathogen. Namely, the results of thisstudy suggest that the effectiveness of a PRRS MLV virus vaccine is not,as it is commonly believed, only determined by its genetic similarity tothe challenge virus, but is also influenced by how it is produced. Theresults of this study are significant in demonstrating that porcinecells are useful in generating an effective MLV vaccine against PRRSvirus.

Abstract. A porcine alveolar macrophage cell line, designated ZMAC-1,was generated and its utility to manufacture an effective PRRS modifiedlive virus (MLV) vaccine was examined. This cell line was found to be100% susceptible to infection by PRRS virus, as evidenced by thesuccessful immunofluorescence staining for viral proteins at 20 hr afterinfection. Moreover, based on multiple step growth curve analyses, thefirst round of PRRS virus replication was determined to be completed by9 hr after infection and the yield of virus progeny during the secondround of replication at 19 hr after infection.

To compare the efficacy of stocks of the MLV vaccine Prime Pac PRRS(Schering-Plough Animal Health) prepared in either ZMAC-1 or the simiancell line MARC-145, a standard immunization-challenge study wasconducted. Six 8 week-old pigs were initially vaccinated intramuscularlywith an equivalent dose (10⁴ TCID₅₀) of the Prime Pac vaccine grown ineither ZMAC-1 or MARC-145 cells, while two additional groups of threeanimals were not immunized. All of these animals, as well as one of thetwo groups of unvaccinated controls, were challenged 4 weeks later with10⁴ TCID₅₀ of the “atypical PRRS abortion storm” virus isolate NADC-20.While the unvaccinated animals experienced an average body weight (BW)loss of −5±4 lb by 7 days after the virulent virus challenge, the PRRSvirus-naïve controls had gained on average 19.7±6 lb during this timeinterval. In contrast, at 7 days post-challenge, the animals vaccinatedwith the MLV virus grown in either ZMAC-1 or MARC-145 cells exhibitaverage BW gains of 8.2±5.2 and 9.3±3.6, respectively. Thus,statistically the Prime Pac MLV vaccine grown in either cell line wasequally effective at reducing the negative effect of the exposure ofpigs to a highly virulent PRRS virus on their growth. Remarkably,analyses of the virus load in serum and lung lavage samples from PRRSvirus-immunized and challenged animals revealed that the vaccine virusgrown in ZMAC-1 cells was significantly (P=0.015) more effective atreducing the extent of viremia at 7 days post-challenge and also ateliminating virulent virus from their lungs by 10 days post-challenge.The observation that the type of cell line used to grow the PRRS MLVvaccine can improve the level of protective immunity elicited by thisproduct against a genetically divergent virulent PRRS virus hassignificant implications for developing a highly effective vaccineagainst this pathogen. Namely, the results of this study suggest thatthe effectiveness of a PRRS MLV virus vaccine is not only, as it iscommonly believed, determined by its genetic similarity to the challengevirus, but is also influenced by how it is produced.

Objectives of this study, at least in part, included the determinationof (1) the growth characteristics of attenuated PRRS virus strains inthe porcine macrophage cell line ZMAC-1; and (2) the immunogencity andefficacy of a PRRS modified live virus vaccine produced in the porcinemacrophage cell line ZMAC-1.

Materials and Methods

Cells. Porcine alveolar macrophage cells were selected, e.g., as furtherdescribed herein, from the lung lavage of a 58 day-old fetus obtainedfrom an SPF (specific pathogen free) sow. The cells were cultured inRPMI-1640 medium, supplemented with 10% fetal bovine serum, sodiumpyruvate and non-essential amino acids, and maintained at 37° C. in a 5%CO₂ atmosphere. A cell line, designated ZMAC-1, was established in Mayof 2006 and has since been continuously growing. Stocks representativeof different temporal subdivisions of this cell line have beencryo-preserved.

Virus. The MLV vaccine Prime Pac PRRS (Schering-Plough Animal Health)was sequentially passaged three times in ZMAC-1 cells. Although thetiter was only 10⁴ TCID₅₀/ml after the first two passages, the virusappeared to have adapted during the third passage as the titer hadincreased to 10^(6.25) TCID₅₀/ml. Accordingly, this progeny was used forvaccination. Stocks of the Prime Pac vaccine were also prepared inMARC-145 cells as previously described (Osorio et al., 2006) andattained a titer of 10⁶ TCID₅₀/ml. The “atypical PRRS abortion storm”virus isolate NADC-20 (Harms et al., 2001) was grown in ZMAC-1 cells andexhibited a maximum titer of 10⁷⁶ TCID₅₀/ml.

Vaccination and Challenge Study: Eighteen 8-week-old SPF pigs (free ofall major swine pathogens including PRRS virus, mycoplasma andcircovirus) were randomly allocated into 6 isolation cubicles (3 pigsper cubicle) at a suite in the Biocontainment Facility at the Universityof Illinois. Animals from four cubicles were injected once in the rumparea with a 2 ml solution containing 10⁴ TCID₅₀ of Prime Pac virus grownin either ZMAC-1 or MARC-145 cells, for a total of 2 cubicles pervaccine formulation (6 pigs total). The six remaining pigs in the twoother cubicles were not immunized and served as unvaccinated controls.Four weeks after vaccination, all of the immunized animals as well asthree of the control pigs housed in one cubicle were challenged with 10⁴TCID₅₀ of PRRS virus strain NADC-20. While the unvaccinated andchallenged animals served to establish the severity of infection by theNADC-20 PRRS virus, the PRRS virus-naïve pigs in the remaining cubiclewere used to provide normal clinical parameters of growth and health.The degree of protective immunity elicited by the vaccine was determinedbased on a comparison of body weight (BW) changes and the appearance ofdepression and respiratory signs. These parameters were monitored dailyfor ten days after the challenge. The level of viremia was determined at0, 4, 7 and 10 days after challenge by measuring infectious units inMARC-145 cells. Ten days after the challenge, the animals wereeuthanized and the viral load in the bronchoalveolar lavage (BAL) fluidwas determined by using real time PCR and virological methods aspreviously described (Zuckermann et al., 2007).

Statistical Analysis. Analysis of Variance was used to determinesignificant differences among groups of pigs in regard to weight gain.Group means were compared by Fisher's least significant differenceprocedure utilizing Stat View software (SAS). To minimize the effect ofBW weight differences between animals at the time of challenge, the datawas calculated as the difference between the BW of the animals at thetime of challenge and 7 days later.

Results

Objective 1. Determine the growth characteristics of attenuated PRRSvirus strains in the porcine macrophage cell line ZMAC-1. The goal ofthis aim was to ascertain the robustness of the ZMAC-1 cell line and itssusceptibility to infection by PRRS virus. In addition, we sought todetermine optimal conditions for the growth of attenuated PRRS virus inthe ZMAC-1 cells and to demonstrate that stocks of MLV vaccine preparedin this cell line could be used for commercial purposes.

Characterization of the porcine alveolar macrophage cell line ZMAC-1.The ZMAC-1 macrophage line has exhibited a very robust growth patternwith a doubling time of approximately 72 hours (FIG. B1; thiscorresponds to FIG. 4A). We have been able to adapt the cells to begrown in 75 cm² flasks and to keep this type of cell culture incontinuous production for the last 15 months. To date, we have generatedmore than 1 billion cells from an initial starting population of a fewthousand. In order to ensure the perpetuity of this valuable cell linemore than 100 frozen cells stocks have been prepared. Every lot has atleast 10 vials, and each vial contains at least 2-3 million cells. Uponthawing a representative vial of every lot, we have determined thatthese vials have >90% viable cells which exhibit vigorous growth within4 days after re-initiation of their culturing. This cell line has beenconfirmed to be of swine origin by the reactivity of 100% of the cellsin the population with the monoclonal antibody K252.1E4, which isspecific for porcine CD45 (Schnitzlein and Zuckermann, 1998; Zuckermannet al., 2001). In addition, the ZMAC-1 cells express the following cellsurface markers: CD14, CD163, CD172, MHC class II, whose presence ischaracteristic of macrophages (FIG. 2).

FIG. B1 (corresponds to FIG. 4A). Growth kinetics of the ZMAC-1 cellline. Established cultures of ZMAC-1 cells in 75 cm² flasks containing2-3.2×10⁵ cells/ml medium were combined with an equal volume of freshmedium to achieve a cell density of 1-1.6×10⁵ cells/ml. Cells werecounted at day 0, 3 and 6 days after fresh medium was delivered into theculture flask.

Growth of PRRS virus in ZMAC-1 cells. The ZMAC-1 cell line is 100%susceptible to infection by PRRS virus, as evidenced by the successfulimmunofluorescence staining for viral proteins at 20 hr after infection(FIG. B2; corresponds to FIG. 5A). Moreover, we have determined that thefirst round of replication of PRRS virus is completed by 9 hr afterinfection (data not shown) and that the peak yield of virus progeny isachieved by the second round of replication at 19 hr after infection(FIG. B3; corresponds to FIG. 4B).

FIG. B2 (corresponds to FIG. 5A). Expression of PRRS virus nucleocapsidprotein in ZMAC-1 cells. At 20 hr after infection with PRRS virus strainNADC-20 at MOI of 1, the cells were fixed and stained with FITC-labeled,anti-PRRS virus nucleocapsid mAb SDOW17.

FIG. B3 (corresponds to FIG. 4B). Multiple step growth curves of PRRSvirus in ZMAC-1 cells. A suspension of ZMAC-1 cells at 1×10⁶/ml wasinfected at an MOI of 0.02 with the attenuated PRRS virus isolate PrimePac. After a one-hr incubation at 37° C. the inoculum was removed bycentrifugation and the cells suspended in medium at a concentration of1×10⁶/ml. One-tenth ml aliquots were removed at the indicated timesafter infection and used for determining the presence of infectiousvirus (TCID₅₀ in MARC-145 cells). Objective 2. Determine theimmunogencity and efficacy of a PRRS modified live virus vaccineproduced in the porcine macrophage cell line ZMAC-1. The goal of thisaim was to compare the levels of protective immunity elicited by thesame PRRS MLV vaccine, in this case the Prime Pac PRRS virus, producedin either the ZMAC-1 cell line or in MARC-145 cell line.

To test the efficacy of the of the Prime Pac PRRS virus vaccine, thathad been prepared in either ZMAC-1 or MARC-145 cells, groups of pigswere either immunized with vaccine virus derived from one of the twocell lines or mock-treated. At the time of challenge, 4 weeks afterimmunization, the average body weight (BW) of all 18 pigs in the studywas 117±8.6 lb. Since no significant differences between the average BWof vaccinated and non-vaccinated animals was observed, vaccination withthe MLV propagated in either ZMAC-1 or MARC-145 cells had no obviousimpact on animal growth. In contrast, at 7 days after challenge with thevirulent NADC-20 isolate, the unvaccinated pigs had an average BW lossof −5±4 lb while the unchallenged animals gained on average 19.7±6 lb(FIG. 6). An average BW gain of 8.2±5.2 and 9.3±3.6 lb, that were notstatistically different from each other, were also noted for the groupsthat had previously received the MLV virus vaccine prepared in eitherZMAC-1 or MARC-145 cells, respectively. Thus, regardless of the type ofcell used to propagate the virus, the Prime Pac MLV vaccine was equallyeffective at diminishing the negative effect of the exposure to virulentPRRS virus on growth, as evidenced by the significant BW loss of PRRSvirus-naïve pigs that had been exposed to a heterologous virulent virus7 days earlier. Remarkably, analyses of the virus load in serum and lunglavage samples from PRRS virus-immunized and challenged animals revealedthat the vaccine virus grown in ZMAC-1 cells was significantly (P=0.015)more effective at reducing the extent of viremia at 7 dayspost-challenge (FIG. 7) and also at eliminating virulent virus fromtheir lungs by 10 days post-challenge (FIG. 8).

FIG. 6. Weight change in pigs after exposure to wild-type PRRS virus.The body weights of PRRS virus-naïve (n=3) and immunized (n=6 for eachtype of cell-generated vaccine) animals were measured immediately priorto and at 7 days after challenge with the wild-type PRRS virus isolateNADC-20. Measurements were also made at these time points for theunchallenged, PRRS virus-naïve animals (n=3). Changes in weight duringthe 7-day interval were averaged for members of each group and thesevalues±the standard error are shown. An asterisk (*) Indicates that thegroup mean is statistically different (P<0.01) from the challenged, PRRSvirus-naïve control animals. Two asterisks (**) are used to indicatethat the group mean is statistically different (P<0.01) from theunchallenged, PRRS virus-naïve control animals.

FIG. 7. Extent and frequency of viremia in pigs after exposure towild-type PRRS virus. Serum samples were collected from PRRS virus-naïveand immunized animals immediately prior to and at the indicated daysafter challenge with the wild-type PRRS virus isolate NADC-20. Samplingswere also taken at these time points for the unchallenged, PRRSvirus-naïve animals. The level of virus load in the serum was determinedby performing titration in MARC-145 cells and then averaged. The datarepresents the average level of viremia for each group. The ratio nextto the symbols indicates the number of viremic pigs (numerator) and thetotal number of pigs per group (denominator).

FIG. 8. Virus load in the bronchalveolar lavage of pigs after exposureto wild-type PRRS virus. Bronchoalveolar lavage was collected from thelungs of PRRS virus-naïve and previously immunized pigs at 10 days afterchallenge with the wild-type PRRS virus isolate NADC-20. Samples werealso obtained at this time from unchallenged, PRRS virus-naïve animals.The level of virus load in the BAL of each animal was determined byperforming titration in MARC-145 cells.

Discussion

The observation that the cell line used to grow the same PRRS MLVvaccine strain can improve the level of protective immunity elicited bythis product against a genetically divergent virulent PRRS virus hasimportant implications for developing a highly effective vaccine againstthis pathogen. Namely, the results of this study suggest that theeffectiveness of a PRRS MLV virus vaccine is not, as it is commonlybelieved, only determined by its genetic similarity to the challengevirus, but is also influenced by how it is produced. A reasonableinterpretation for the observations described above is that thebiological properties of the vaccine virus were modified in a positiveway by simply being grown in ZMAC-1 cells. Consequently, a moreeffective protective immune response developed in the vaccinatedanimals. Thus, the results of this study demonstrate that a moreeffective MLV vaccine against PRRS virus can be created.

REFERENCES FOR EXAMPLE 5

Harms, P. A., Sorden, S. D., Halbur, P. G., Bolin, S. R., Lager, K. M.,Morozov, I., Paul, P. S. 2001. Experimental reproduction of severedisease in CD/CD pigs concurrently infected with type 2 porcinecircovirus and porcine reproductive and respiratory syndrome virus. VetPathol. 38:528-39.

Hill, H., M. JA, K. J J, H. A, and R. C. 2004. PRRS Control: To Hell andBack. American Assoc Swine Veterinarians, Des Moines, Iowa, USA, Mar.6-9, 2004:369-376.

Mengeling, W. L., K. M. Lager, and A. C. Vorwald. 1999. Safety andefficacy of vaccination of pregnant gilts against porcine reproductiveand respiratory syndrome. Am J Vet Res 60:796-801.

Mengeling, W. L., A. C. Vorwald, K. M. Lager, and S. L. Brockmeier.1996. Comparison among strains of porcine reproductive and respiratorysyndrome virus for their ability to cause reproductive failure. Am J VetRes 57:834-9.

Osorio, F. A., F. Zuckermann, R. Wills, W. Meier, S. Christian, J.Galeota, and A. Doster. 1998. PRRSV: comparison of commercial vaccinesin their ability to induce protection against current PRRSV strains ofhigh virulence. Allen D. Leman Swine Conference 25:176-182.

Kwon, B., Ansari, I. H., Osorio, F. A, Pattnaik, A. K. 2006. Infectiousclone-derived viruses from virulent and vaccine strains of porcinereproductive and respiratory syndrome virus mimic biological propertiesof their parental viruses in a pregnant sow model. Vaccine. 24:7071-80.

Schnitzlein, W. M., Zuckermann, F. A. 1998. Determination of thespecificity of CD45 and CD45R monoclonal antibodies through the use oftransfected hamster cells producing individual porcine CD45 isoforms.Vet Immunol Immunopathol. 60:389-401.

Zuckermann, F. A., Schnitzlein, W. M., Thacker, E., Sinkora, J.,Haverson, K. 2001. Characterization of monoclonal antibodies assigned tothe CD45 subgroup of the Third International Swine CD Workshop. VetImmunol Immunopathol. 80:165-74.

Zuckermann, F. A, Alvarez Garcia, E., Diaz Luque, I.,Christopher-Hennings, J., Doster, A., Brito, M., Osorio, F. 2007.Assessment of the efficacy of commercial porcine reproductive andrespiratory syndrome virus (PRRSV) vaccines based on measurement ofserologic response, frequency of gamma-IFN-producing cells andvirological parameters of protection upon challenge. Vet Microbiol.123:69-85.

EXAMPLE 6 Cell Materials and Growth of Cells

Cell materials. Cells designated as ZMAC-1 were prepared and depositedwith a recognized International Depositary Authority, the American TypeCulture Collection (ATCC; 10801 University Boulevard, Manassas, Va.,United States of America) under the Budapest Treaty. The accessionnumber is ATCC Patent Deposit No. PTA-8764 for cells characterized asbeing of Sus scrofa (pig/swine) lung tissue origin. According to theATCC Certificate of Deposit document dated Dec. 7, 2007 (Budapest TreatyOn The International Recognition Of The Deposit Of Microorganisms ForThe Purposes Of Patent Procedure; International Form; Receipt In TheCase Of An Original Deposit Issued Pursuant To Rule 7.3 And ViabilityStatement Issued Pursuant To Rule 10.2), the Date of Receipt of Cultureis Nov. 14, 2007 for ATCC® Patent Deposit Designation PTA-8764.

Growth of cells. In embodiments of the invention, cells such as ZMAC-1cells are cultivated in vitro. In general, principles of mammalian cellculture are applied. For example, cells can be cultured in the presenceof antibiotics; gentamicin is used but not necessarily required.

Cells are prepared as follows (culture medium: RPMI-1640 supplementedwith 10% fetal bovine serum, sodium pyruvate (1 mM; Mediatech Cellgro,Cat. No. 25-000-C1), non-essential amino acids (1×; Mediatech CellgroCat. No. 25-025-C1), and Gentamicin (50 mcg/ml; Gibco, Cat. No.15750-060). The cells are maintained at cell concentrations of about 1to 5×10e5 per ml. These cells generally grow in suspension. Discretecolonies of loosely adherent cells can develop but most of the cellswill be growing in suspension. Normal cultures will produce floatingcell clumps. To reduce adherence, the preferred type of culture flask isSarstedt Tissue Culture Flask for suspension cell with PE vented cap(Cat. No. 83.1813.502). Established flasks can be harvested every 4-5days with removal of ⅔ of the fluid and addition of fresh culturemedium. New flasks are established by adding at least 3-6 million cellsin a 20 ml volume in a T25 flask (minimum of 1.5×10e5 cells/ml). Growthcan be enhanced by adding 2-10 ng/ml of macrophage colony-stimulatingfactor (mouse; Sigma-Aldrich Product No. M9170). Cell freezing can beaccomplished by mixing equal volumes of ice-cold suspensions of cells at4-8 million cells per ml and ice-cold freezing medium (90% serum, 10%DMSO). Chilled cryovials are filled with the cells suspended in freezingmedium and maintained at ice-cold temperature during the process.

FIG. 9 illustrates growth of ZMAC-1 cells in the presence of growthfactors. ZMAC-1 cells at 1.2×10⁵ cell per ml, were cultured for 8 dayswithout exogenous growth factor or in the presence of the indicatedconcentration of either macrophage colony stimulating factor (MCSF) orgranulocyte-macrophage colony stimulating factor (GMCSF). The cellconcentration was determined with the aid of a hemocytometer at thefourth and eighth day of culture. The y-axis indicates cells/ml (×10⁵).

In an embodiment, a cell of the invention is represented by a celldesignated as ZMAC-1 or a deposit with the American Type CultureCollection designated as ATCC Patent Deposit No. PTA-8764 or derivedtherefrom.

EXAMPLE 7 Generation of Cell Materials and Methods of Isolation fromFetal Porcine Samples

Further compositions and methods were developed. In an independentattempt, materials were derived from a sow at 60 days of gestation fromthe swine herd at the University of Illinois Veterinary MedicineResearch Farm (identified as Sow number 5850). Following euthanasia, theuterus was aseptically removed from the abdominal cavity and transportedto the cell culture laboratory. Manipulations were generally performedusing a bio-safety cabinet under sterile conditions. Six fetuses wereaseptically harvested from the uterus and placed in plastic Petridishes. Lung organs, with the trachea intact and attached to the lung,was dissected away from the heart, esophagus and other membranes. Theoutside of the lung was thoroughly rinsed with sterile Hank's balancedsalt solution (HBSS) to remove any visible blood and other contaminatingremaining tissue. Cells in the airways of the lungs from each of the 6fetuses were isolated separately by bronchoalveolar lavage by placingthe lung in a clean and sterile Petri dish and filling the airways with10 ml of sterile HBSS. The 10 ml of HBSS was propelled into the lungwith the aid of a 10 cc syringe and a 1 inch 18 g needle, which wasinserted through the lumen of the trachea. The fluid was gentlypropelled through the trachea while constraining it via compression withforceps to prevent backflow of the HBSS, resulting in the lungs becomingvisibly inflated with the fluid. Afterwards the fluid containing thelung lavage cells was self-expelled from the lung by simply releasingthe tracheal compression. The cell suspension collected in the Petridish was transferred to a sterile 15 ml conical plastic tube, and wasunderlayed with 3-4 ml of warm Ficoll-Hypaque 1077. Immediatelyafterwards cell suspension was purified via isopycnic centrifugation(400 g for 30 minutes at room temperature).

In an embodiment, cells can be purified by isopycnic centrifugationusing Ficoll-Hypaque 1077 either the day of isolation or at a latertime, e.g., 1, 2, or 3 weeks later. It is recognized that thispurification procedure can facilitate removal of certain components suchas red cells, and potentially other material, present in an originalpreparation. The band of cells obtained after centrifugation at theinterface between the Ficoll-Hypaque 1077 and medium was harvested andwashed with HBSS twice and the cells recovered each time bycentrifugation. After the second centrifugation, the recovered cellpellet from each fetal lung lavage was suspended in culture medium:RPMI-1640 supplemented with 10% fetal bovine serum, sodium pyruvate (1mM; Mediatech Cellgro, Cat. No. 25-000-C1), non-essential amino acids(1×; Mediatech Cellgro Cat. No. 25-025-C1), and placed independently inone well of 6-well plate (Sarstedt) for suspension culture. Each wellwas labeled from 1-6 to identify the cells purified independently fromeach of the six fetuses. Although in this attempt very few and verysmall cells (<100) were recovered after the isopycnic centrifugationprocedure, within 14 days after establishment of the initial cultures,significant growth was observed in every well. Since the macrophageprogenitors harvested from the fetal lung were apparently very small andcould have a density greater than 1.077 (thus going through the densitymedium to the bottom of the tube during the isopycnic centrifugation),in the case of fetus #1, the red cell pellet obtained after theisopycnic centrifugation was also harvested and placed in culture andlabeled P1. In this case, although the predominant cell type at theinitiation of the culture consisted of red blood cells, a small numberof very small mononuclear cells was observed. At 16 and 24 days afterthe initiation of the culture the growth in cells derived from the lunglavage of fetus #4 exhibited sufficient growth to merit splitting intonew wells of a 6 well plate. Cells derived from this culture were namedZMAC1107-4. Similarly, growth in the well labeled as P1, which wasderived from the red blood cell pellet was clearly evident at 36 daysafter the initiation of the culture and was also split into 2 wells.Cells derived from this culture were named ZMAC1107-P1. The growth ofcells was evident by the presence of cell clusters comprised of 2, 4, 8,16 or more cells per cluster.

At 37 days after the initiation of the culture of the ZMAC1107-4, theculture medium for this cell line was supplemented in one of duplicatewells with 10 ng/ml of macrophage colony stimulating factor (mouse;Sigma-Aldrich Product No. M9170). Seven days later it was clear that thegrowth of the ZMAC1107-4 cells had been significantly aided at thisearly stage of culture by the exogenous supplementation with the growthfactor, and thereafter the medium of all cultures was supplemented withMCSF at 5-10 ng/ml. Cultures are fed every 4-6 days by removing byaspiration half the volume of the cell culture and replacing it withfresh medium supplemented with the growth factor. The robust growth ofboth the ZMAC 1107-4 and ZMAC 1107-P1 lines was evident by the formationof cell colonies growing in suspension and loosely attached to theculture surface.

In another independent attempt, further compositions and methods weredeveloped. A sow identified as number 9093, at 54 days of gestation wasobtained from the swine herd at the University of Illinois VeterinaryMedicine Research Farm. Following euthanasia, the uterus was asepticallyremoved from the abdominal cavity and transported to the cell culturelaboratory. All manipulations from this point forward were done insideof a bio-safety cabinet under sterile conditions. Eight fetuses wereaseptically harvested from the uterus and placed in plastic Petri dishesand their lungs, with the trachea intact and attached to the lung,dissected away from the heart, esophagus and other membranes. Theoutside of the lung was thoroughly rinsed with HBSS to remove anyvisible blood and other contaminating remaining tissue. Cells in theairways of the lungs from each of the eight fetuses were isolatedseparately by bronchoalveolar lavage by placing the lung in a clean andsterile Petri dish and filling the airways with 10 ml of sterile Hank'sbalanced salt solution (HBSS). The 10 ml of HBSS was propelled into thelung with the aid of a 10 cc syringe and a 1 inch 18 g needle, which wasinserted through the lumen of the trachea. The fluid was gentlypropelled through the trachea while compressing it with forceps toprevent backflow of the HBSS, resulting in the lungs becoming visiblyinflated with the fluid. Afterwards the fluid containing the lung lavagecells was self-expelled from the lung by simply reducing compression ofthe trachea. In some cases the lung was gently pressed down with theblunt end of scissors to help expel the remaining lavage fluid. The cellsuspension collected in the Petri dish was transferred to a sterile 15ml conical plastic tube, and centrifuged for 10 minutes at 1,500 RPM ina table top clinical centrifuge. The recovered cell pellet from eachfetal lung lavage was suspended in culture medium: RPMI-1640supplemented with 10% fetal bovine serum, sodium pyruvate (1 mM;Mediatech Cellgro, Cat. No. 25-000-C1), non-essential amino acids (1×;Mediatech Cellgro Cat. No. 25-025-C1) and placed independently in onewell of 6-well plate (Sarstedt) for suspension culture. Each well waslabeled from 1-8 to identify the cells purified independently from eachof the eight fetuses. Approximately 10,000 cells were initially place inculture from each fetal lung lavage. Growth in these cultures wasevident by 5 days. Large clumps of cells were evident in culturesderived from fetuses #1, 3, 6, 7 and 8.

At 12 days after the initiation of the culture the non-adherent andloosely adherent cells from all 8 fetal lung lavage cell cultures wereharvested by gently pipetting and purified by isopycnic centrifugationusing Ficoll-Hypaque 1077. This was accomplished by transferring thecell suspension to a sterile 15 ml conical plastic tube, which wasunderlay with 3-4 ml of warm Ficoll-Hypaque 1077 and then centrifuged at400 g for 30 minutes at room temperature. A clearly visible band ofcells was obtained after centrifugation at the interface between theFicoll-Hypaque 1077. Medium was harvested and washed with HBSS twice,and the cells were recovered each time by centrifugation. After thesecond centrifugation, the recovered cell pellet from each individualfetal lung lavage culture was suspended in 3 ml of culture medium:RPMI-1640 supplemented with 10% fetal bovine serum, sodium pyruvate (1mM; Mediatech Cellgro, Cat. No. 25-000-C1), non-essential amino acids(1×; Mediatech Cellgro Cat. No. 25-025-C1), and placed independently inone well of 6-well plates (Sarstedt) for suspension culture and labeled1-8, which corresponded directly to the original labeling of thecultures. Five days later all cell cultures were fed 2 cc of freshculture medium. Nine days later significant growth of cells insuspension as well as adherent cells was evident in cultures derivedfrom fetal lung lavage cell cultures labeled 3 and 6, which exhibited asignificant number of macrophage colonies growing in suspension as wellas loosely adherent round macrophages. A number of large sphericalsyncytial cells, surrounded by small macrophages forming a structureappearing as a cellular crown, were observed. At 26 days after thebeginning of the culture, the cell cultures were fed fresh mediumsupplemented with 5 ng/ml of MCSF (mouse; Sigma-Aldrich Product No.M9170). Five days later vigorous growth of cell macrophage coloniesgrowing in suspension as well as loosely adherent on the surface of theculture plate was observed in cultures derived from fetuses #3, #6 and#8. These lines were named ZMAC1207-3, ZMAC1207-6, and ZMAC1207-8respectively and were expanded a few days later by transferring to T75flasks in culture medium supplemented with 5-10 ng/ml of MCSF.

STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS

All references mentioned throughout this application, for example patentdocuments including issued or granted patents or equivalents; patentapplication publications; unpublished patent applications; andnon-patent literature documents or other source material; are herebyincorporated by reference herein in their entireties, as thoughindividually incorporated by reference. In the event of anyinconsistency between cited references and the disclosure of the presentapplication, the disclosure herein takes precedence. Some referencesprovided herein are incorporated by reference to provide information,e.g., details concerning sources of starting materials, additionalstarting materials, additional reagents, additional methods ofsynthesis, additional methods of analysis, additional biologicalmaterials, additional cells, and additional uses of the invention.

All patents and publications mentioned herein are indicative of thelevels of skill of those skilled in the art to which the inventionpertains. References cited herein can indicate the state of the art asof their publication or filing date, and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed herein, it should be understood that compounds knownand available in the art prior to Applicant's invention, includingcompounds for which an enabling disclosure is provided in the referencescited herein, are not intended to be included in the composition ofmatter claims herein.

Any appendix or appendices hereto are incorporated by reference as partof the specification and/or drawings.

Where the terms “comprise”, “comprises”, “comprised”, or “comprising”are used herein, they are to be interpreted as specifying the presenceof the stated features, integers, steps, or components referred to, butnot to preclude the presence or addition of one or more other feature,integer, step, component, or group thereof. Thus as used herein,comprising is synonymous with including, containing, having, orcharacterized by, and is inclusive or open-ended. As used herein,“consisting of” excludes any element, step, or ingredient, etc. notspecified in the claim description. As used herein, “consistingessentially of” does not exclude materials or steps that do notmaterially affect the basic and novel characteristics of the claim(e.g., relating to the active ingredient). In each instance herein anyof the terms “comprising”, “consisting essentially of” and “consistingof” may be replaced with either of the other two terms, therebydisclosing separate embodiments and/or scopes which are not necessarilycoextensive. The invention illustratively described herein suitably maybe practiced in the absence of any element or elements or limitation orlimitations not specifically disclosed herein.

Whenever a range is disclosed herein, e.g., a temperature range, timerange, composition or concentration range, or other value range, etc.,all intermediate ranges and subranges as well as all individual valuesincluded in the ranges given are intended to be included in thedisclosure. This invention is not to be limited by the embodimentsdisclosed, including any shown in the drawings or exemplified in thespecification, which are given by way of example or illustration and notof limitation. It will be understood that any subranges or individualvalues in a range or subrange that are included in the descriptionherein can be excluded from the claims herein.

The invention has been described with reference to various specificand/or preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention. It will beapparent to one of ordinary skill in the art that compositions, methods,devices, device elements, materials, procedures and techniques otherthan those specifically described herein can be employed in the practiceof the invention as broadly disclosed herein without resort to undueexperimentation; this can extend, for example, to starting materials,biological materials, reagents, synthetic methods, purification methods,analytical methods, assay methods, and biological methods other thanthose specifically exemplified. All art-known functional equivalents ofthe foregoing (e.g., compositions, methods, devices, device elements,materials, procedures and techniques, etc.) described herein areintended to be encompassed by this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by embodiments, preferred embodiments, andoptional features, modification and variation of the concepts hereindisclosed may be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

REFERENCES

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I claim:
 1. A method of isolating a cell from a porcine fetal lung,comprising the steps of providing a porcine fetal subject; obtaining acell-containing sample from said subject; separating a cell from saidsample, and culturing the sell for at least 5 passages, wherein the cellis capable of replicating porcine reproductive and respiratory syndrome(PRRS) virus; thereby isolating said cell.
 2. A method of isolating acell from a porcine fetal lung, comprising the steps of providing aporcine fetal subject; obtaining a cell-containing sample from saidsubject, wherein said cell-containing sample is a bronchoalveolar lavagesample; and separating a cell from said sample, wherein the cell iscapable of replicating PRRS virus; thereby isolating said cell.
 3. Themethod of claim 1, wherein the separating step is by density gradientcentrifugation.
 4. The method of claim 1 wherein the cell is amacrophage.
 5. The method of claim 1, further comprising growing thecell for at least 10 days in continuous culture.
 6. The method of claim1, wherein the culturing is for at least 10, 20, or 50 passages.
 7. Themethod of claim 1, wherein the porcine fetal subject is from about 30 toabout 90 days of gestational age.
 8. A method of isolating a cell from aporcine fetal lung, comprising the step of providing a porcine fetalsubject; obtaining a cell-containing sample from said subject,separating a cell from said sample, and subcloning the cell, wherein thecell is capable of replicating PRRS virus; thereby isolating said cell.9. The method of claim 1, further comprising culturing the cell in agrowth medium comprising a growth factor composition.
 10. The method ofclaim 9, wherein the growth factor composition comprises macrophagecolony stimulating factor (MCSF) or granulocyte-macrophage colonystimulating factor (GMCSF).
 11. The method of claim 9, wherein thegrowth factor composition comprises MCSF at a concentration of 5 to 10ng/mL.
 12. The method of claim 2, wherein the cell is a macrophage. 13.The method of claim 2, further comprising the step of culturing the cellfor at least 5 passages.
 14. The method of claim 2, wherein the porcinefetal subject is from about 30 to about 90 days of gestational age. 15.The method of claim 2, further comprising culturing the cell in a growthmedium comprising a growth factor composition of MCSF or GMCSF.
 16. Themethod of claim 8, wherein the cell is a macrophage.
 17. The method ofclaim 8, further comprising the step of culturing the cell for at least5 passages.
 18. The method of claim 8, wherein the porcine fetal subjectis from about 30 to about 90 days of gestational age.
 19. The method ofclaim 8, further comprising culturing the cell in a growth mediumcomprising a growth factor composition of MCSF or GMCSF.
 20. The methodof claim 4, wherein the macrophage is an alveolar macrophage.